Pathogen reduction of fresh plasma using riboflavin and ultraviolet light: effects on plasma coagulation proteins.

BACKGROUND: Treatment with riboflavin and ultraviolet (UV) light reduces the pathogens present in blood components. This study assessed changes to the coagulation proteins that had occurred during this treatment of fresh plasma units before freezing. STUDY DESIGN AND METHODS: Twenty fresh plasma units (230 +/- 30 mL) were treated by the Mirasol process (CaridianBCT Biotechnologies) and frozen within 8 hours of donation. Plasma units were combined with 35 mL of a 500 micromol/L riboflavin solution in an illumination bag to achieve a final concentration of approximately 60 micromol/L riboflavin. The bag was placed in the Mirasol illuminator and exposed to UV light (6.24 J/mL). Samples were frozen before and after treatment. RESULTS: Recoveries observed were 67.7 +/- 3.9% Factor (F)XI, 68.5 +/- 3.3% FVIII:C, 78.8 +/- 4.5% fibrinogen, 78.9 +/- 4.1% FV, 79.0 +/- 4.2% FVII, 79.0 +/- 8.6% F IX, 79.7 +/- 2.6% FX, and 85.0 +/- 3.7% FII. Von Willebrand factor (VWF) antigen, VWF:ristocetin cofactor, and ADAMTS13 recoveries were 87.0 +/- 7.1, 85.5 +/- 6.6, and 73.3 +/- 15.2%, respectively, while that of protein C was 83.6 +/- 2.6%. A loss of high-molecular-weight VWF multimers was observed in most units. Recoveries for protein S, antithrombin, and plasmin inhibitor were greater than 90%. The mean FVIII:C concentration, after treatment, was 0.76 +/- 0.17 IU/mL. CONCLUSIONS: As with other pathogen reduction technologies, the Mirasol process resulted in some loss of coagulation factor activity. For most Mirasol-treated units and for most of the tested factors this is unlikely to have clinical impact, but trials are required to demonstrate this.

Freezing of buffy coat-derived, leukoreduced platelet concentrates in 6 percent dimethyl sulfoxide.

BACKGROUND: There has recently been renewed interest in freezing platelets (PLTs) in dimethyl sulfoxide (DMSO) for the treatment of major traumatic injuries, especially in military situations. This study examined PLTs that were frozen in small volumes of 6 percent DMSO at -80 degrees C. STUDY DESIGN AND METHODS: Buffy coat-derived pooled leukoreduced PLT concentrates were frozen in 6 percent DMSO and stored at -80 degrees C. Assays included hypotonic shock response (HSR); aggregation; glycoprotein (GP)Ibalpha and P-selectin binding sites; annexin V binding to phosphatidylserine, glycocalicin, and lactate dehydrogenase (LDH). Cone and plate technology (DiaMed Impact-R, DiaMed) was used to test PLT function under near physiologic conditions. RESULTS: The freeze-thaw loss of PLTs was 23 percent. HSR was 17 +/- 7 percent. Cytometry demonstrated two populations of PLTs: one with normal levels of GPIbalpha binding sites (27 x 10(3) +/- 3 x 10(3)/PLT) and one with reduced levels (5.5 x 10(3) +/- 1.2 x 10(3)/PLT). There were 1.4 x 10(3) +/- 0.2 x 10(3) P-selectin binding sites per PLT. Annexin V binding to phosphatidylserine was 50 +/- 9 percent and LDH was 496 +/- 207 IU per 10(12) PLTs. Surface coverage and aggregate size, as measured by the DiaMed Impact-R, were similar to those observed with PLTs stored for 2 days at 22 degrees C. CONCLUSION: Some degree of activation was demonstrated by the proportion of PLTs with reduced levels of GPIbalpha binding sites, increased P-selectin expression, and increased Annexin V binding. LDH concentrations indicated a degree of lysis. The DiaMed Impact-R results showed that the PLTs were still capable of adhering to surfaces and forming aggregates under shear force.

The effects of leukodepletion on the generation and removal of microvesicles and prion protein in blood components.

BACKGROUND: Universal leukodepletion (LD) has been implemented in the United Kingdom to reduce the risk of transfusion-transmitted variant Creutzfeldt-Jakob disease. If LD causes microvesiculation of blood cells, however, potentially infectious membrane-associated prion could reach the final products. STUDY DESIGN AND METHODS: We have measured microvesicles (MV) derived from red cells (RBC-MV), platelets (PLT-MV), and white blood cells (WBC-MV) and cellular prion protein (PrP(c)) in blood components produced by four whole-blood, five RBC, three PLT, and two plasma LD filters and three plateletpheresis techniques. RESULTS: RBC-MV and PLT-MV were either unaltered or reduced by all processes, with PLT-MV reduced 10-fold by RBC LD and greater than 300-fold by plasma LD. WBC-MV were reduced or unchanged by RBC and PLT LD and reduced by plasma LD. Whole-blood filtration appeared to increase MVs derived from granulocytes, but the load in the final components was comparable to that in processed RBCs in additive solution. PrP(c) was reduced by whole-blood, RBC, and plasma LD and unchanged by PLT techniques. There were differences between various filters and techniques, which were generally minor compared to the overall effects. CONCLUSION: These findings suggest no detrimental effects of LD processes in terms of generation of MVs or PrP(c) release.

The expression of prion protein by endothelial cells: a source of the plasma form of prion protein?

The neuronal prion protein (PrPC) is also expressed within peripheral tissues including human blood. The majority of blood PrPC is found within the plasma fraction. We hypothesized that the vascular endothelium could be a source of this PrPC. Reverse transcription polymerase chain reaction demonstrated that both human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1) expressed PrPC mRNA. Flow cytometry confirmed PrPC expression on HMEC-1s and HUVECs (120900 +/- 15058 and 58327 +/- 4577 molecules PrPC/cell respectively), with no upregulation following cellular activation. Confocal immunofluorescence microscopy confirmed that HMEC-1s and HUVECs were positive for PrPC on the plasma membrane. Time-resolved dissociation-enhanced fluoroimmunoassay (DELFIA) analysis of cell culture medium demonstrated a slow constitutive release of soluble PrPC not associated with activation. In contrast to von Willebrand factor antigen, PrPC plasma levels in vivo decrease following desmopressin therapy in patients with von Willebrand disease. Measurement of PrPC plasma levels in patients with varying blood counts demonstrated no association between cell count and PrPC concentration. However, there was a higher level of PrPC in plasma from patients with end-stage renal failure. In conclusion, endothelial cells of both macrovascular and microvascular origin expressed high levels of PrPC which can be constitutively released into the cell culture medium.

The effect of methylene blue photoinactivation and methylene blue removal on the quality of fresh-frozen plasma.

BACKGROUND: T: he effects of using fresh or frozen-thawed plasma, WBC reduction of plasma before freezing, and the use of two different methylene blue (MB) removal filters on the quality of MB-treated plasma were compared. STUDY DESIGN AND METHODS: In a paired study (n = 11/arm) plasma was frozen within 8 hours of collection, thawed, MB photoinactivated, and then filtered using one of two MB removal filters. Fresh plasma (n = 16) and plasma WBC reduced before freezing (n = 19) were MB inactivated. RESULTS: Freeze-thawing resulted in loss of activity of FXII and VWF of 0.06 and 0.04 units per mL, respectively, but no significant loss of activity of factors II through XI or fibrinogen. Further loss of activity occurred after MB treatment: FII (0.07 IU/mL), FV (0.11 U/mL), FVII (0.08 IU/mL), FVIII (0.28 IU/mL), F IX (0.12 IU/mL), FX (0.16 IU/mL), FXI (0.28 U/mL), FXII (0.15 U/mL), VWF antigen (0.05 IU/mL), VWF activity (0.06 U/mL), and fibrinogen (0.79 g/L). Losses due to this step were significantly (5-10%) lower in fresh plasma compared to frozen-thawed plasma. Neither MB removal filter resulted in significant loss of activity of any factor studied. CONCLUSION: MB removal, by either of the available filters, has little impact on the coagulation factor content of plasma, but freezing of plasma before MB treatment results in a small additional loss.